Delivery of agents to inflamed tissues using folate-targeted liposomes

ABSTRACT

The invention described herein pertains to folate-receptor targeted agents comprising therapeutic agents useful for the treatment of inflammatory disease, including folate-receptor targeted liposomes (folate-targeted liposomes) containing entrapped therapeutic agents and folate-receptor targeted dendrimers conjugated to therapeutic agents (folate-targeted dendrimer conjugates), useful for the treatment of inflammatory disease, including auto-immune disease, as well as to folate-targeted liposomes containing entrapped imaging agents and dendrimer conjugates conjugated to imaging agents, for use in the diagnosis and monitoring of treatment in such disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/349,434, filed 28 May 2010, which is incorporated by referenceherein.

TECHNICAL FIELD

The invention described herein pertains to folate-receptor targetedagents comprising therapeutic agents useful for the treatment ofinflammatory disease, including folate-receptor targeted liposomes(folate-targeted liposomes) containing entrapped therapeutic agents andfolate-receptor targeted dendrimers conjugated to therapeutic agents(folate-targeted dendrimer conjugates), useful for the treatment ofinflammatory disease, including auto-immune disease, as well as tofolate-targeted liposomes containing entrapped imaging agents anddendrimer conjugates conjugated to imaging agents, for use in thediagnosis and monitoring of treatment in such disease.

BACKGROUND AND SUMMARY OF THE INVENTION

Liposomes are closed, spherical vesicles comprising amphiphilic lipidssuch as phospholipids, sphingolipids, and/or other lipids, includingsterols such as cholesterol and cholesterol ester salts, in proportionssuch that they arrange themselves into multiple concentric bilayers whenhydrated in aqueous solutions. Using any of a number of methods, suchliposomes can be converted into single bilayer liposomes. These singlebilayer liposomes are useful carriers both of hydrophilic (lipophobic)agents, which can reside entrapped in the aqueous interior of theliposome, and of hydrophobic (lipophilic) agents, which can resideentrapped in the lipid bilayer. The utility of liposomes as carriers fortherapeutic agents has been recognized and has lead to the developmentof long-circulating liposomes. By altering the composition of the lipidbilayer in such liposomes by, for example, the inclusion of a surfacecoat of flexible biocompatible hydrophilic chains, the resultingliposomes have improved stability. The surface coat serves to protectthe liposome from uptake by organs of the mononuclear phagocyte system,especially the liver, lung and spleen, and from the plasma componentswhich are involved in liposome uptake. One example of the hydrophilicchain constituent of the surface coat is polyethylene glycol (PEG), inwhich the terminal hydroxy group may be capped with a methyl group toform a methyl ether group (denoted as mPEG or MPEG). (Unlessspecifically defined otherwise, in this specification PEG will includethe hydroxy terminated form, the methyl ether terminated form, and anyother similarly terminated form in the context of the surface coat of aliposome.)

Inflamed tissue regions in the body have been imaged usingfolate-targeted imaging agents. For example, the folate-receptortargeted, radionuclide conjugate imaging agent EC20 (folate-Tc99m), hasbeen used to image rheumatoid arthritis sites in vivo.

In WO 94/07466 there is disclosed a PEG coated liposomal compositioncontaining an entrapped therapeutic agent for concentrating therapeuticsin an inflamed tissue region in the body. The beneficial effects of theliposomes of WO 94/07466 (EP-0662820) are questioned in WO 03/105805,which discloses PEG-coated liposomes composed of non-chargedvesicle-forming lipids, optionally containing not more than 5 molpercent of charged vesicle-forming lipids, and containing a watersoluble corticosteroid for the site-specific treatment of inflammatorydisorders.

In a folate receptor positive mouse model of ovarian cancer,folate-targeted liposomes carrying the entrapped fluorescent dye calceinhave been shown to unload the dye in ovarian cancer cells andtumor-asssociated macrophages within tumor ascites fluid associated withan intraperitoneal cancer both ex vivo and after in vivo administrationand collection of the ascites fluid for analysis. (M. J. Turk et al.,Cancer Letters 213 (2004) 165-172.) The same reference discloses folatetargeted liposomes in which the lipid phase includes tritiatedcholesterol-oleoyl ether.

Disclosed herein are folate-targeted liposomal compositions comprisingan anti-inflammatory agent as an entrapped agent, pharmaceuticalcompositions comprising such liposomal compositions, and the use of suchcompositions in the treatment of an inflammatory disease, such as in aninflamed tissue region in the body. Entrapped agents can be encapsulatedwithin the aqueous interior of the liposomes and/or dissolved into thehydrocarbon regions of their bilayers. Also disclosed arefolate-targeted liposomal compositions comprising an imaging orvisualizing agent as an entrapped agent, as well as the use of suchliposomal compositions for diagnosis or monitoring the treatment of aninflammatory disease in an inflamed tissue region in the body.

Further disclosed herein are dendrimers conjugated to active agents(folate-targeted dendrimer conjugates), such as anti-inflammatoryagents, pharmaceutical compositions comprising such dendrimerconjugates, and the use of such compositions in the treatment of diseasesuch as inflammatory disease. Dendrimers are known classes of compoundswhich are generally regarded as repeatedly branched, approximatelyspherical molecules and are sometimes referred to as arborols or cascademolecules. Also, disclosed are folate-targeted dendrimer conjugatecompositions comprising an imaging or visualizing agent, as well as theuse of such compositions for diagnosis or monitoring the treatment of adisease such as inflammatory disease in an inflamed tissue region in thebody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (top) shows the relative biodistribution of uptake of³H-cholesterol labeled folate-targeted liposomes in the aortas (1) andhearts (2) of Normal Chow (N) and Western Diet (W) mice; thebiodistribution in a number of organs (aorta (1), heart (2), liver (3),kidney (4), spleen (5), intestine (6), muscle (7), skin (8) and lung(9)) is shown in FIG. 1 (bottom).

FIG. 2 shows whole body imaging of the atherosclerotic sites in Apo E KOmice fed a western diet for one week using a folate-targeted liposomalcomposition, with the entrapped fluorescent dye DiD, preinjection (top)and 2 hours post injection (bottom) in the absence (left) and presence(right) of pre-administered folic acid. The scale shows low (bottom) tohigh (top) intensity of fluorescence.

FIG. 3 shows the imaging of the atherosclerotic sites in the aortic archin Apo E KO mice fed a western diet for one week using a folate-targetedliposomal composition, with the entrapped fluorescent dye DiD.Atherosclerotic plaques showed a 5-fold increase in mean fluorescence.

FIG. 4 shows whole body imaging of the atherosclerotic sites in Apo E KOmice fed a western diet for four weeks before imaging under fourconditions: (A) control with no imaging agent, (B) DiD-entrappedfolate-targeted liposomal composition, (C) non-targeted DiD-entrappedliposomal composition, and (D) DiD-entrapped folate-targeted liposomalcomposition administered with an excess of folic acid.

FIG. 5 shows radiographic (X-ray) imaging at preinjection (0) and 2, 4,24 and 48 hours post injection of an Apo E KO mouse dosed with aniobitridol-entrapped folate-targeted liposomal composition.

FIG. 6 shows radioisotopic imaging in Apo E KO mice fed a western dietfor four weeks before dosing using a ⁹⁹Tc-entrapped folate-targetedliposomal composition, administered either alone (A) or with an excessof folic acid (B).

FIG. 7 shows radioisotopic imaging of the whole animal (top) and excisedhearts (bottom) using the folate-receptor targeted, radionuclideconjugate imaging agent EC20 (folate-Tc99m) in Apo E KO mice fed awestern diet for three weeks and dosed as (A) a control (Untreated)group which received a folate-targeted liposomal composition whichcontained only phospate buffered saline as the entrapped agent, (B) agroup (NT Liposome) which received a non-targeted liposomal compositionwhich contained betamethasone as the entrapped agent, and (C) a group(Fol-Liposome) which received a folate-targeted liposomal compositionwhich contained betamethasone as the entrapped agent.

FIG. 8 shows the relative EC20 heart uptake of the groups of FIG. 7.

FIG. 9 shows the biodistribution of EC20 uptake in the organs (heart(1), lung (2), liver (3), spleen (4), kidney (5), muscle (6), skin (7)and intestine (8) of the groups of FIG. 7.

FIG. 10 shows radioisotopic imaging (top) of the hearts (1) and aortas(8) and the biodistribution (bottom) of the organs (heart (1), lung (2),liver (3), intestine (4), kidney (5), spleen (6), muscle (7), aorta (8)and skin(9)) using the folate-receptor targeted, radionuclide conjugateimaging agent EC20 (folate-Tc99m) in Apo E KO mice fed a western dietfor 4 weeks and dosed as (A) a control (Untreated) group which receiveda folate-targeted liposomal composition which contained only phospatebuffered saline as the entrapped agent, (B) a group (NT Liposome) whichreceived a non-targeted liposomal composition which containedbetamethasone as the entrapped agent, and (C) a group (Fol-Liposome)which received a folate-targeted liposomal composition which containedbetamethasone as the entrapped agent.

FIG. 11 shows the imaging of the intestines of mice in an intestinalinflammation model using imaging with (A) a DiD-entrappedfolate-targeted liposomal composition, (B) a DiD-entrappedfolate-targeted liposomal composition administered with an excess offolic acid, and (C) a non-targeted DiD-entrapped liposomal composition.

FIG. 12 shows the imaging of the paws of rats in an arthritis modelusing imaging with (A) a DiD-entrapped folate-targeted liposomalcomposition, (B) a DiD-entrapped folate-targeted liposomal compositionadministered with an excess of folic acid, and (C) a non-targetedDiD-entrapped liposomal composition.

FIG. 13 shows the imaging of mice in an inflammatory muscle injury modelshowing a healthy mouse imaged with a folate-targeted DiD-entrappedliposomal composition (Healthy), a cardiotoxin treated mouse imaged witha non-targeted DiD-entrapped liposomal composition (NT Liposome), and acardiotoxin treated mouse imaged with a folate-targeted DiD-entrappedliposomal composition (Fol Liposome). The scale shows low (left) to high(right) intensity of uptake of the label.

FIG. 14 shows the cell bound fluorescence measured by flow cytometry inthioglycolate recruited macrophages for control non-targetedcalcein-entrapped liposomal compositions (shaded curve), and forfolate-targeted calcein-entrapped liposomal compositions in the presence(middle curve) and absence (right curve) of excess free folic acid.

FIG. 15 shows the imaging of the paws of rats in an arthritis modelshowing a diseased rat imaged with a non-targeted DiD-entrappedliposomal composition (NT Liposome), a diseased rat imaged with afolate-targeted DiD-entrapped liposomal composition (Fol-Liposome), anda healthy rat with a folate-targeted DiD-entrapped liposomal composition(Healthy).

FIG. 16 shows the imaging in mice implanted with (M109) tumors whichexpress the folate receptor at high levels on the surface andconcomitantly used in the muscle injury model or the ulcerative colitismodel when imaged with a folate-targeted DiD-entrapped liposomalcomposition (Fol-Liposome).

FIG. 17 shows the imaging in mice implanted with (M109) tumors whichexpress the folate receptor at high levels on the surface andconcomitantly used in the muscle injury model when imaged with anon-targeted DiD-entrapped liposomal composition (NT Liposome), with afolate-targeted DiD-entrapped liposomal composition (Fol-Liposome), andwith empty folate-targeted liposome (injected empty folate-targetedliposome first, then dosed with a folate-targeted DiD-entrappedliposomal composition) (Competition with Folate Liposome).

FIG. 18 shows the imaging in mice implanted with (M109) tumors whichexpress the folate receptor at high levels on the surface andconcomitantly used in an intestinal inflammation model when imaged witha non-targeted DiD-entrapped liposomal composition (NT Liposome), with afolate-targeted DiD-entrapped liposomal composition (Fol-Liposome), andwith empty folate-targeted liposome (injected empty folate-targetedliposome first, then dosed with a folate-targeted DiD-entrappedliposomal composition) (Fol-Liposome with Competition).

FIG. 19 shows schematically the types of dendrimer conjugates used inthe imaging studies.

FIG. 20 shows a synthetic scheme for the preparation of afolate-targeted dendrimer conjugate conjugated to a dye. Folate-targeteddendrimer conjugates conjugated to active agents, such asanti-inflammatory agents, may be prepared similarly.

FIG. 21 shows imaging of cells which express the folate receptor at highlevels on the surface (control) and in the presence of a folate-targeteddendrimer conjugate conjugated to FITC dye (FolDendFITC) alone and inthe presence of excess folic acid (FolDendFITC w Excess Folic Acid).

FIG. 22 shows whole body imaging of the atherosclerotic sites in Apo EKO mice fed a western diet with a folate-targeted dendrimer conjugateconjugated to Cy5.5 dye (FolDend(G3)Cy5.5), with a non-targeteddendrimer conjugate conjugated to Cy5.5 dye, and in competition with afolate-targeted dendrimer conjugate which lacks a conjugated dye(FolDend(G3)Cy5.5 W Competion).

FIG. 23 shows the imaging of the atherosclerotic sites in the aorticarch in Apo E KO mice fed a western diet with a folate-targeteddendrimer conjugate conjugated to Cy5.5 dye (FolDend(G3)Cy5.5), with anon-targeted dendrimer conjugate conjugated to Cy5.5 dye, and incompetition with a folate-targeted dendrimer conjugate which lacks aconjugated dye (FolDend(G3)Cy5.5 W Competion).

FIG. 24 shows the imaging of (the intestines of) mice in an intestinalinflammation model using imaging with a folate-targeted dendrimerconjugate conjugated to Cy5.5 dye (FolDend(G3)Cy5.5), with anon-targeted dendrimer conjugate conjugated to Cy5.5 dye, and incompetition with a folate-targeted dendrimer conjugate which lacks aconjugated dye (FolDend(G3)Cy5.5 W Competion).

DETAILED DESCRIPTION

In one embodiment, there is provided a folate-receptor targeted agentcomprising a therapeutic agent useful for the treatment of inflammatorydisease. In one embodiment, the folate-receptor targeted agent is afolate-targeted liposomal composition comprising an anti-inflammatoryagent as an entrapped agent. In one embodiment, the folate-receptortargeted agent is a folate-targeted dendrimer conjugate comprising afolate-targeted dendrimer conjugated to an anti-inflammatory agent.

In one embodiment, there is provided a folate-targeted liposomalcomposition comprising an anti-inflammatory agent as an entrapped agent.In general, the embodiment of the liposome may be as described herein orof any conventional composition and may be constructed as describedherein or by any conventional methodology with the folate-targetingligand attached as described herein or by any conventional linkagestructure or methodology.

The lipid components used in forming the embodiments of the liposomesmay be any of the variety of vesicle-forming lipids, includingphospholipids, sphingolipids, and/or other lipids, including sterolssuch as cholesterol and cholesterol ester salts. Suitable lipids for theembodiments include those having two hydrocarbon chains, typically acylchains, and a polar head group, such as phospholipids and glycolipids.As used herein, unless otherwise defined or clear from the context,phospholipid includes any one phospholipid or combination ofphospholipids capable of forming liposomes. Phospholipids includephosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidicacid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where thetwo hydrocarbon chains are typically between about 14-22 carbons inlength, and have varying degrees of unsaturation. The glycolipidsinclude cerebrosides and gangliosides. Phosphatidylcholines, includingthose obtained from natural sources or those that are partially orwholly synthetic, or of variable chain length and unsaturation, aresuitable. Phospholipids which contain saturated alkyl chains, yielding arelatively high transition temperature, are suitable for theembodiments. These include, for example,distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylcholine(DSPC) and hydrogenated soy phosphatidylcholine (HSPC). One embodimentof the liposomal composition disclosed herein is one wherein the lipidbilayer is primarily composed of DSPC/cholesterol with a mol ratio ofabout 56:40.

The folate-targeting components used in forming the embodiments of theliposomes may be any of those known in the preparation offolate-targeted liposomes or those described herein. For examples of thepreparation of folate-targeted liposomal compositions, see, for example,R. J. Lee et al., J. Biological Chemistry, 269(5) (1994) 3198-3204; andR. J. Lee et al., Biochimica et Biophysica Acta, 1233 (1995) 134-144.

One embodiment is a liposomal composition as described herein whereinthe lipid bilayer comprises a comprises a folate targeting conjugatecomposed of (a) a lipid having a polar head group and a hydrophobictail, (b) a hydrophilic polymer having a first end and a second end,said polymer attached at its first end to the head group of the lipid,and (c) a folate ligand (Fol) attached to the second end of the polymer,and wherein the folate ligand is a folic acid residue or an analog orderivative thereof. Folic acid analogs or derivatives thereof includethe (unnatural) D-isomer of folic acid and derivatives other relatedfolic acid receptor binding molecules, such as folinic acid, pteroicacid, pteropolyglutamic acid, receptor-binding pteridines such astetrahydropterins, dihydrofolates, tetrahydrofolates, and their deazaand dideaza analogs. The terms “deaza” and “dideaza” analogs refer tothe art-recognized folate analogs having a carbon atom substituted forone or two nitrogen atoms in the naturally occurring folic acidstructure. Other folate analogs or derivatives include the folatereceptor-binding analogs aminopterin, amethopterin (methotrexate), andderivatives thereof.

For example, in one embodiment, the hydrophilic polymer of the folatetargeting conjugate is a PEG having an average molecular weight of about200-5000 in which the end groups, prior to attachment, are independentlyamino, hydroxy, thiol or carboxy. In one embodiment the hydrophilicpolymer of the folate targeting conjugate is a PEG having an averagemolecular weight of about 3200-3400 in which the end groups, prior toattachment, are independently amino or thiol. In one embodiment thehydrophilic polymer of the folate targeting conjugate is a PEG having anaverage molecular weight of about 3200-3400 in which the end groups,prior to attachment, are each amino, such as for example, NH₂PEG₃₂₀₀NH₂or NH₂PEG₃₄₀₀NH₂, which also may be denoted as a polyoxyethylenebis-amine or a PEG bis-amine, such as for example PEG bis-amine,M_(r)˜3350. In another embodiment, the hydrophilic polymer of the folatetargeting conjugate is a PEG having an average molecular weight of about3200-3400 in which the end groups, prior to attachment, are amino andthiol, which also may be denoted amino-PEG-SH. An amino-PEG-SH may beobtained from the corresponding PEG bis-amine by a conventional method,such as by the addition of Traut's reagent.

In one embodiment, the folate-targeted liposomal composition, the lipidhaving a polar head group and a hydrophobic tail, is a phosphatidylgroup as defined herein. In one embodiment, the phosphatidyl groupcomprises two saturated acyl groups each of which is between about 14-22carbons in length. In one embodiment, the phosphatidyl group comprisestwo saturated acyl groups each of which is between about 16-18 carbonsin length. In one embodiment, the phosphatidyl group is a distearoyl(DS) derivative.

In the folate targeting conjugate the polar head group of the lipid isattached to the first end of the hydrophilic polymer by any conventionalmethod. In one embodiment, the polar head group is part of aphosphatidylethanolamine, wherein the phosphatidyl group has any of themeanings herein, and the amino group of the ethanolamine portion islinked to an amino group of the first end of the hydrophilic polymer bya bis-acyl group, such as a succinyl group. A succinyl group also may bedenoted as a 1,4-dioxobutane-1,4-diyl group. In another embodiment, thepolar head group is part of a phosphatidylethanolamine, wherein thephosphatidyl group has any of the meanings herein, and the amino groupof the ethanolamine portion is linked to a thiol group of the first endof the hydrophilic polymer via addition of the thiol group to anN-maleiimidocaproylphospatidylethanolamine. In one embodiment of any ofthe above, the phosphatidylethanolamine isdistearoylphosphatidylethanolamine (DSPE).

In one embodiment of the folate-targeted liposomal composition, thefolate ligand is a folic acid residue which is attached to an aminogroup at the second end of the hydrophilic polymer by acylation with thea-carbonyl group or the γ-carbonyl group of the glutamic acid portion offolic acid. In another embodiment of the folate-targeted liposomalcomposition, the folate ligand is a folic acid residue which is attachedto an amino group at the second end of the hydrophilic polymer byacylation with the γ-carbonyl group of the glutamic acid portion offolic acid.

In one embodiment of the folate-targeted liposomal composition, thefolate targeting conjugate comprises distearoylphosphatidylethanolaminelinked to the first end of a bis-amino PEG and a folic acid residuewhich is attached to the amino group at the second end of the bis-aminoPEG by acylation with the γ-carbonyl group of the glutamic acid portionof folic acid. In a further embodiment, thedistearoylphosphatidylethanolamine is linked to the first end of thebis-amino PEG by a succinyl group.

A further embodiment of the above liposomal composition is one whereinthe folate targeting conjugate comprisesdistearoylphosphatidylethanolamine wherein the amino group of theethanolamine portion is linked by a succinyl group to the amino group ofthe first end of a PEG having an average molecular weight of about3200-3400 in which the end groups, prior to attachment, are each amino,and wherein the folate ligand is a folic acid residue which is attachedto the amino group at the second end of the PEG by acylation with theγ-carbonyl group of the glutamic acid portion of folic acid.

In one embodiment of the folate-targeted liposomal composition, asdescribed herein, the folate targeting conjugate is present at about0.01 mol % to about 1 mol % in the lipid bilayer. In another embodiment,the folate targeting conjugate is present at about 0.05 mol % to about0.5 mol % in the lipid bilayer. In another embodiment, the folatetargeting conjugate is present at about 0.1 mol % in the lipid bilayer.

One embodiment is a liposomal composition as described herein whereinthe lipid bilayer further comprises a hydrophilic coating composed of(a) a lipid having a polar head group and a hydrophobic tail and (b) ahydrophilic polymer having a first end and a second end, said polymerattached at its first end to the head group of the lipid and optionallycapped at its second end. The hydrophilic coating components used informing the embodiments of the liposomes may be any of those known inthe preparation of hydrophilic coated liposomes or those describedherein.

In general, the lipid of the hydrophilic coating having a polar headgroup and a hydrophobic tail can be any of those disclosed herein forthe preparation of a folate targeting conjugate. Thus, in oneembodiment, the lipid having a polar head group and a hydrophobic tailis a phosphatidyl group as defined herein. In one embodiment, thephosphatidyl group comprises two saturated acyl groups each of which isbetween about 14-22 carbons in length. In one embodiment, thephosphatidyl group comprises two saturated acyl groups each of which isbetween about 16-18 carbons in length. In one embodiment, thephosphatidyl group is a distearoyl (DS) derivative.

The hydrophilic polymer of the hydrophilic coating can be any of anumber of biocompatible hydrophilic polymers. In one embodiment, thehydrophilic polymer is a polyethylene glycol (PEG), a polylactic acid(PLA), a polyglycolic acid (PGA) or a polyvinyl alcohol (PVA), as wellas copolymers of lactic and glycolic acids, in which the first endgroup, prior to attachment, is amino, hydroxy or thiol. The hydrophilicpolymer is optionally capped with methyl, ethyl, or another unreactivegroup. In another embodiment, the hydrophilic polymer is a polyethyleneglycol with an average molecular weight of about 2000, which is uncappedor capped with a methyl or ethyl group.

The polar head group of the lipid is attached to the first end of thehydrophilic polymer of the hydrophilic coating by any conventionalmethod. In one embodiment, the polar head group is part of aphosphatidylethanolamine, wherein the phosphatidyl group has any of themeanings herein, and the amino group of the ethanolamine portion islinked directly to a carbon of the first end of the hydrophilic polymer,or via a linker to an amino, hydroxy or thiol group of the first end ofthe hydrophilic polymer by a linking group. The amino group of theethanolamine portion may be linked directly to a carbon of the first endof the hydrophilic polymer, for example by converting a hydroxy group toa leaving group, such as a triflate, and alkylating the amino group ofthe ethanolamine. The amino group of the ethanolamine portion of aphosphatidylethanolamine may be linked to a hydroxy group of the firstend of the hydrophilic polymer, for example, by a linking group such asa carbonyl group or a 4-chloro-1,3,5-triazinyl group; or the amino groupof the ethanolamine portion may be linked to an amino group of the firstend of the hydrophilic polymer, for example, by a linking group such asa carbonyl group, a succinyl group or a 4-chloro-1,3,5-triazinyl group.The amino group of the ethanolamine portion of aphosphatidylethanolamine may be linked to a thiol group of the first endof the hydrophilic polymer by formation of a disulfide bond between analkyl thiol group on the amino group of the ethanolamine and the thiolgroup of the hydrophilic polymer. For the preparation of a hydrophiliccoating in which the hydrophilic polymer is not capped, the group at thesecond end may be protected using a conventional protecting group whilethe first end is attached to the polar head group of the lipid of thehydrophilic coating having a polar head group and a hydrophobic tail;then the protecting group is removed. For example, a hydroxy group atthe second end of the hydrophilic polymer may be protected using atrimethylsilyl group, which is removed following the attachment of thefirst end of the hydrophilic polymer to the polar head group. Oneembodiment is a liposomal composition as described herein wherein thelipid bilayer further comprises a hydrophilic coating comprisingdistearoylphosphatidyl-ethanolamine and polyethylenegycol 2000(PEG₂₀₀₀)or methoxypolyethyleneglycol 200 (mPEG₂₀₀₀).

One embodiment is a liposomal composition as described herein whereinthe hydrophilic coating is present at about 1 to 20 mol % in the lipidbilayer. Another embodiment is a liposomal composition as describedherein wherein the hydrophilic coating is present at about 4 mol % inthe lipid bilayer.

One embodiment is a liposomal composition as described herein whereinthe liposome bilayer comprises a mol ratio ofDSPC/cholesterol/mPEG2000-DSPE of about 56:40:4. Another embodiment is aliposomal composition as described herein wherein the liposome bilayercomprises a mol ratio of DSPC/Chol/mPEG₂₀₀₀-DSPE/Fol-PEG₃₄₀₀DSPE ofabout 56:40:4:0.1.

One embodiment is a liposomal composition as described herein whereinthe average particle size of the liposome is about 30 to 200 nM. Anotherembodiment is a liposomal composition as described herein wherein theaverage particle size of the liposome is about 40 to 120 nM. A furtherembodiment is a liposomal composition as described herein wherein theaverage particle size of the liposome is about 50 to 100 nM.

A further embodiment of folate-targeted liposomal compositions, asdescribed herein, is the ability to deliver more of the entrapped agentto an inflamed site than corresponding non-targeted liposomalcompositions, which are the same as the folate-targeted liposomalcompositions, except the folate targeting conjugate, such asfolate-PEG-DSPE, is omitted.

One embodiment is a liposomal composition, or folate-targeted dendrimerconjugate, as described herein wherein the entrapped anti-inflammatoryagent is an anti-inflammatory steroid. In one embodiment, theanti-inflammatory steroid is a sytemically administered (lipophilic)anti-inflammatory steroid. In one embodiment, the anti-inflammatorysteroid is betamethasone, dexamethasone, flumethasone,methylprednisolone, paramethasone, prednisolone, prednisone,triamcinolone, hydrocortisone or cortisone. A further embodiment is aliposomal composition as described herein wherein the entrapped agent isbetamethasone.

In another embodiment, the anti-inflammatory steroid is a topicallyadministered anti-inflammatory steroid. In this sense, topicallyadministered includes administration by any of a number of nonsystemicmeans, including by inhalation, suppository, topical cream ointment,foam, lotion or gel, etc. In one embodiment, the anti-inflammatorysteroid is alcomethasone dipropionate, amcinonide, betamethasonedipropionate, betamethasone monopropionate, betamethasone 17-valerate,budesonide, budesonide disodium phosphate, ciclomethasone,clobetasol-17-propionate, clobetasone-17-butyrate, cortisone acetate,deprodone propionate, desonide, desoxymethasone, dexamethasone acetate,diflucortolone valerate, diflurasone diacetate, diflucortolone,difluprednate, flumetasone pivalate, flunisolide, fluocinolone acetonideacetate, fluocinonide, fluocortolone, fluocortolone caproate,fluocortolone hexanoate, fluocortolone pivalate, fluormetholone acetate,fluprednidene acetate, fluticasone propionate, halcinonide,halometasone, hydrocortisone acetate, hydrocortisone-17-butyrate,hydrocortisone-17-valerate, medrysone, methylprednisolone acetate,mometasone furoate, parametasone acetate, prednicarbate, prednisoloneacetate, prednylidene, rimexolone, tixocortol pivalate, triamcinoloneacetonide, triamcinolone alcohol or triamcinolone hexacetonide. In oneembodiment, the anti-inflammatory steroid is budesonide, flunisolide orfluticasone propionate.

In another embodiment, the anti-inflammatory steroid is a water solubleanti-inflammatory steroid. Another embodiment is a liposomal compositionas described herein wherein the anti-inflammatory steroid isbetamethasone sodium phosphate, desonide sodium phosphate, dexamethasonesodium phosphate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, cortisone sodium phosphate, cortisone sodium succinate,methylprednisolone disodium phosphate, methylprednisolone sodiumsuccinate, methylprednisone disodium phosphate, methylprednisone sodiumsuccinate, prednisolone sodium phosphate, prednisolone sodium succinate,prednisone sodium phosphate, prednisone sodium succinate, prednisolamatehydrochloride, triamcinolone acetonide disodium phosphate ortriamcinolone acetonide dipotassium phosphate. In one embodiment, theanti-inflammatory steroid is budesonide disodium phosphate.

One embodiment is a liposomal composition, or folate-targeted dendrimerconjugate, as described herein wherein the entrapped anti-inflammatoryagent is a non-steroidal anti-inflammatory drug (NSAID), which also maybe denoted as a non-steroidal anti-inflammatory agent (NSAIA) or as anon-steroidal anti-inflammatory medicine (NSAIM). In one embodiment, theNSAID comprises a propionic acid derivative such as, for example,ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen or oxaprozin.In one embodiment, the NSAID comprises an acetic acid derivative, suchas, for example, indomethacin, sulindac, etodolac or diclofenac. In oneembodiment, the NSAID comprises an oxicam derivative, such as, forexample, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam orisoxicam. In one embodiment, the NSAID comprises a fenamic acidderivative, such as, for example, mefenamic acid, meclofenamic acid,flufenamic acid or tolfenamic acid. In one embodiment, the NSAIDcomprises a selective COX-2 (cyclooxygenase-2) inhibitor (coxib), suchas, for example, celecoxib, rofecoxib, valdecoxib, parecoxib,lumiracoxib or etoricoxib.

Another embodiment is a liposomal composition, or folate-targeteddendrimer conjugate, as described herein wherein the entrappedanti-inflammatory agent comprises another drug useful in the treatmentof rheumatoid arthritis or other autoimmune disease including anantiproliferative, immunomodulator or immunosuppresant agent,comprising, for example, aspirin, methotrexate, sulfasalazine,D-penicillamine, nambumetone, aurothioglucose, auranofin, othergold-containing compound, colloidal gold, cyclosporin, tacrolimus,pimecrolimus or sirolimus.

Because liposomes are useful carriers both of hydrophilic, lipophobicagents, which can reside entrapped in the aqueous interior of theliposome, and of hydrophobic, lipophilic agents, which can resideentrapped in the lipid bilayer, it is possible to load a lipsome asdisclosed herein simultaneously with more than one anti-inflammatoryagent, any of which may be hydrophilic or lipophilic. Similarly, it ispossible to load a lipsome as disclosed herein simultaneously with ananti-inflammatory agent and a further therapeutic agent, any of whichmay be hydrophilic or lipophilic. Thus, one embodiment is a liposomalcomposition as described herein comprising more than oneanti-inflammatory agent as an entrapped agent. Another embodiment is aliposomal composition as described herein wherein there are twoentrapped anti-inflammatory agents. A further embodiment is a liposomalcomposition as described herein comprising an anti-inflammatory agentand a further therapeutic agent.

As a further embodiment, there is provided a pharmaceutical compositioncomprising a liposomal composition comprising an anti-inflammatory agentas an entrapped agent, or folate-targeted dendrimer conjugate, asdescribed herein and further comprising at least one pharmaceuticallyacceptable carrier or excipient. The pharmaceutical composition may beformulated in a conventional manner as known for the formulation ofliposomes for administration to patients. For example, thepharmaceutical composition may be a sterile dispersion in a bufferedaqueous vehicle, optionally containing a tonicity adjusting agent.Alternatively, for example, the pharmaceutical composition may be asterile, non-pyrogenic lyophilized product comprising a buffer and/or atonicity adjusting agent, which is suitable for reconstitution withsterile water for injection to form a sterile dispersion. In eithercase, the sterile dispersion may diluted in 5% dextrose to anappropriate concentration prior to intravenous infusion. Apharmaceutical composition includes a veterinary composition.

As a further embodiment, there is provided a liposomal compositioncomprising an anti-inflammatory agent as an entrapped agent, orfolate-targeted dendrimer conjugate, as described herein for use as amedicament. As a further embodiment, there is provided a liposomalcomposition comprising an anti-inflammatory agent as an entrapped agent,or folate-targeted dendrimer conjugate, as described herein for use inthe treatment of an inflammatory disease. As a further embodiment, thereis provided the use of a liposomal composition comprising ananti-inflammatory agent as an entrapped agent, or folate-targeteddendrimer conjugate, as described herein for the treatment of aninflammatory disease. As a further embodiment, there is provided the useof a liposomal composition comprising an anti-inflammatory agent as anentrapped agent, or folate-targeted dendrimer conjugate, as describedherein for the manufacture of a medicament for the treatment of aninflammatory disease.

As a further embodiment, there is provided a method of treatment of aninflammatory disease in a subject in need thereof, comprisingadministering an effective amount of a liposomal composition comprisingan anti-inflammatory agent as an entrapped agent, or folate-targeteddendrimer conjugate, as described in any of claims. In one embodimentthe subject is a mammal. In one embodiment the subject is a human.

For any of the above uses or methods of treatment, in one embodiment theinflammatory disease comprises an inflamed tissue region in the body. Inone embodiment the use or method is one wherein the inflammatory diseaseis arthritis, arteriosclerosis, graft-versus-host disease, multiplesclerosis, osteomyelitis, psoriasis or inflammatory bowel disease, suchas Crohn's disease or ulcerative colitis. In one embodiment the use ormethod one wherein the inflammatory disease is arthritis. In oneembodiment the use or method one wherein the inflammatory disease isrheumatoid arthritis. In one embodiment the use or method one whereinthe inflammatory disease is osteoarthritis. In one embodiment the use ormethod one wherein the inflammatory disease is arteriosclerosis. In oneembodiment the use or method one wherein the inflammatory disease isatherosclerosis. In one embodiment the use or method one wherein theinflammatory disease is inflammatory bowel disease, such as Crohn'sdisease or ulcerative colitis.

One embodiment is the use of a folate-targeted liposomal composition, orfolate- targeted dendrimer conjugate, comprising an imaging orvisualizing agent as an entrapped agent for diagnosis or monitoring thetreatment of an inflammatory disease in an inflamed tissue region in thebody. Another embodiment is the use of a folate-targeted liposomalcomposition, or folate-targeted dendrimer conjugate, comprising animaging or visualizing agent as an entrapped agent for the manufactureof an agent for diagnosis or monitoring the treatment of an inflammatorydisease in an inflamed tissue region in the body. Another embodiment isan agent for diagnosis or monitoring the treatment of an inflammatorydisease, comprising a folate-targeted liposomal composition, orfolate-targeted dendrimer conjugate, comprising an imaging orvisualizing agent as an entrapped agent. Another embodiment is apharmaceutical composition comprising a folate-targeted liposomalcomposition, or folate-targeted dendrimer conjugate, comprising animaging or visualizing agent as an entrapped agent and furthercomprising at least one pharmaceutically acceptable carrier orexcipient. A further embodiment is a method of using a folate-targetedliposomal composition, or folate-targeted dendrimer conjugate,comprising an imaging or visualizing agent as an entrapped agent fordiagnosis or monitoring the treatment of an inflammatory disease in aninflamed tissue region in the body in a subject in need thereof. Oneembodiment of the method is wherein the subject is a mammal. Anotherembodiment of the method is wherein the subject is a human.

For any use, agent, pharmaceutical composition or method of using afolate-targeted liposomal composition wherein the entrapped agent is afolate-targeted liposomal composition comprising an imaging orvisualizing agent, other than the difference in the entrapped agent,embodiments of the folate-targeted liposomal composition may becharacterized in the same manner as embodiments of a folate-targetedliposomal composition comprising an anti-inflammatory agent as anentrapped agent as described herein. Similarly for folate-targeteddendrimer conjugates comprising an imaging or visualizing agent,embodiments may be characterized in the same manner as embodimentscomprising an anti-inflammatory agent.

One embodiment of the above use, agent, pharmaceutical composition ormethod is one wherein the entrapped or conjugated agent is apharmaceutically acceptable fluorescent dye. In one embodiment, thefluorescent dye is DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indodicarbocyanine perchlorate),calcein or fluorescein.

Another embodiment of the above use, agent, pharmaceutical compositionor method is one wherein the entrapped or conjugated agent is a contrastagent for X-ray, MRI (magnetic resonance imaging) or ultrasound. Oneembodiment is the X-ray contrast agent is iobitridol.

Another embodiment of the above use, agent, pharmaceutical compositionor method is one wherein the entrapped or conjugated agent comprises aradionuclide. In one embodiment, the radionuclide is an isotope ofgallium, indium, copper, technitium or rhenium. In another embodiment,the radionuclide is an isotope of technitium.

One embodiment comprises a kit comprising a folate-targeted liposomalcomposition, or folate-targeted dendrimer conjugate, comprising animaging or visualizing agent as an entrapped agent as described hereinand instructions for diagnosis or monitoring the treatment of aninflammatory disease.

As described above, a folate-targeted liposomal composition comprisingthe fluorescent dye calcein has been described. (M. J. Turk et al.,Cancer Letters 213 (2004) 165-172.) However, a folate-targeted liposomalcomposition comprising an imaging or visualizing agent other than afluorescent dye as an entrapped agent provides a novel embodiment. For afolate-targeted liposomal composition comprising an imaging orvisualizing agent other than a fluorescent dye as an entrapped agent,embodiments of the folate-targeted liposomal composition may becharacterized in the same manner as embodiments of a folate-targetedliposomal composition comprising an anti-inflammatory agent as anentrapped agent as described herein.

In one embodiment the folate-targeted liposomal composition, orfolate-targeted dendrimer conjugate, comprising an imaging orvisualizing agent other than a fluorescent dye as an entrapped agent isone wherein the entrapped agent is an X-ray contrast agent. In oneembodiment, the X-ray contrast agent is iobitridol.

In another embodiment the folate-targeted liposomal composition, orfolate-targeted dendrimer conjugate, comprising an imaging orvisualizing agent other than a fluorescent dye as an entrapped agent isone wherein the entrapped agent comprises a radionuclide. In oneembodiment, the radionuclide is an isotope of gallium, indium, copper,technitium or rhenium. In one embodiment, the radionuclide is an isotopeof technitium.

One embodiment is a pharmaceutical composition comprising a liposomalcomposition, or folate-targeted dendrimer conjugate, comprising animaging or visualizing agent other than a fluorescent dye as anentrapped agent and further comprising at least one pharmaceuticallyacceptable carrier or excipient. Another embodiment is the use of afolate-targeted liposomal composition, or folate-targeted dendrimerconjugate, comprising an imaging or visualizing agent other than afluorescent dye as an entrapped agent for diagnosis or monitoring thetreatment of an inflammatory disease in an inflamed tissue region in thebody. Another embodiment is the use of a folate-targeted liposomalcomposition, or folate-targeted dendrimer conjugate, comprising animaging or visualizing agent other than a fluorescent dye as anentrapped agent for the manufacture of an agent for diagnosis ormonitoring the treatment of an inflammatory disease in an inflamedtissue region in the body. Another embodiment is an agent for diagnosisor monitoring the treatment of an inflammatory disease, comprising afolate-targeted liposomal composition, or folate-targeted dendrimerconjugate, comprising an imaging or visualizing agent other than afluorescent dye as an entrapped agent. A further embodiment is a methodof using a folate-targeted liposomal composition, or folate-targeteddendrimer conjugate, comprising an imaging or visualizing agent otherthan a fluorescent dye as an entrapped agent for diagnosis or monitoringthe treatment of an inflammatory disease in an inflamed tissue region inthe body in a subject in need thereof. One embodiment of the method iswherein the subject is a mammal. Another embodiment of the method iswherein the subject is a human.

An embodiment is a kit comprising a folate-targeted liposomalcomposition, or folate-targeted dendrimer conjugate, comprising ananti-inflammatory agent as an entrapped agent, or a pharmaceuticalcomposition thereof, as described herein, and an imaging agent for theinflammatory condition. In one embodiment of the kit, the imaging agentis a folate-targeted imaging agent. In another embodiment of the kit,the folate-targeted imaging agent is a folate-targeted liposomalcomposition, or folate-targeted dendrimer conjugate, as describedherein. In a further embodiment of the kit, the folate-targeted imagingagent is a folate-targeted conjugate of an imaging agent. In oneembodiment of the kit, the folate-targeted conjugate is EC20(folate-Tc99m).

An additional embodiment of any of the above described kits is a kitfurther comprising instructions for diagnosis or monitoring thetreatment of an inflammatory disease. An additional embodiment of any ofthe above described kits is a kit further comprising instructions forthe treatment of an inflammatory disease.

Embodiments of the invention are further described by the followingenumerated clauses:

1. A folate-receptor targeted agent comprising a therapeutic agentuseful for the treatment of inflammatory disease.

2. The folate-receptor targeted agent of clause 1 which is afolate-targeted liposomal composition comprising an anti-inflammatoryagent as an entrapped agent.

3. The liposomal composition of clause 2 wherein the lipid bilayer isprimarily composed of DSPC/cholesterol with a mol ratio of about 56:40.

4. The liposomal composition of clause 2 or 3 wherein the lipid bilayercomprises a comprises a folate-targeting conjugate composed of (a) alipid having a polar head group and a hydrophobic tail, (b) ahydrophilic polymer having a first end and a second end, said polymerattached at its first end to the head group of the lipid, and (c) afolate ligand (Fol) attached to the second end of the polymer, andwherein the folate ligand is a folic acid residue or an analog orderivative thereof.

5. The liposomal composition of clause 4 wherein the hydrophilic polymerof the folate targeting conjugate is a PEG having an average molecularweight of about 200-5000 in which the end groups, prior to attachment,are independently amino, hydroxy, thiol or carboxy.

6. The liposomal composition of any of clauses 2-5 wherein the folatetargeting conjugate is present at about 0.01 mol % to about 1 mol % inthe lipid bilayer.

7. The liposomal composition of any of clauses 4-6 wherein the lipidbilayer further comprises a hydrophilic coating composed of (a) a lipidhaving a polar head group and a hydrophobic tail and (b) a hydrophilicpolymer having a first end and a second end, said polymer attached atits first end to the head group of the lipid and optionally capped atits second end.

8. The liposomal composition of clause 7 wherein the hydrophilic coatingis a polyethylene glycol (PEG), a polylactic acid (PLA), a polyglycolicacid (PGA) or a polyvinyl alcohol (PVA).

9. The liposomal composition of clause 7 or 8 wherein the hydrophilicpolymer is a polyethylene glycol with an average molecular weight ofabout 2000, which is uncapped or capped with a methyl or ethyl group.

10. The liposomal composition of any of clauses 2-9 wherein theentrapped agent is an anti-inflammatory steroid.

11. The liposomal composition of any of clauses 2-10 wherein theentrapped agent is a drug useful in the treatment of rheumatoidarthritis or other autoimmune disease including an antiproliferative,immunomodulator or immunosuppresant agent.

12. A method of treatment of an inflammatory disease in a subject inneed thereof, comprising administering an effective amount of aliposomal composition as described in any of clauses 2-12.

13. A pharmaceutical composition comprising a liposomal compositioncomprising an anti-inflammatory agent as an entrapped agent as describedin any of clauses 2-12 and further comprising at least onepharmaceutically acceptable carrier or excipient.

14. A composition comprising a folate-targeted liposomal compositioncomprising an imaging or visualizing agent as an entrapped agent andfurther comprising at least one pharmaceutically acceptable carrier orexcipient.

15. A method of using a folate-targeted liposomal composition comprisingan imaging or visualizing agent as an entrapped agent for diagnosis ormonitoring the treatment of an inflammatory disease in an inflamedtissue region in the body in a subject in need thereof.

16. A kit comprising a folate-targeted liposomal composition comprisingan imaging or visualizing agent as an entrapped agent as described inclause 14 and instructions for diagnosis or monitoring the treatment ofan inflammatory disease.

17. A kit comprising a folate-targeted liposomal composition comprisingan anti-inflammatory agent as an entrapped agent, as described in any ofclauses 2-11, or a pharmaceutical composition thereof, as described inclause 13, and an imaging agent for the inflammatory condition.

18. The folate-receptor targeted agent of clause 1 which is afolate-targeted dendrimer conjugate comprising a folate-targeteddendrimer conjugated to an anti-inflammatory agent.

19. The folate-targeted dendrimer conjugate of clause 18 wherein thedendrimer comprises a folate-targeting conjugate wherein thefolate-targeting ligand (Fol) is a folic acid residue or an analog orderivative thereof, a hydrophilic coating, and a conjugatedanti-inflammatory agent.

20. The folate-targeted dendrimer conjugate of clause 19 wherein thefolate targeting conjugate is present at about 12-25% of the dendrimerictermini; the hydrophilic coating residue is present at about 25-40% ofthe dendrimeric termini; and the conjugated to anti-inflammatory agentis present at about 3-13% of the dendrimeric termini.

21. The folate-targeted dendrimer conjugate of clause 19 or 20 whereinthe hydrophilic coating is a polyethylene glycol (PEG), a polylacticacid (PLA), a polyglycolic acid (PGA) or a polyvinyl alcohol (PVA).

22. The liposomal composition of any of clauses 19-21 wherein thehydrophilic polymer is a polyethylene glycol with an average molecularweight of about 2000, which is uncapped or capped with a methyl or ethylgroup.

23. The folate-targeted dendrimer conjugate of any of clauses 19-22wherein the conjugated anti-inflammatory agent is an anti-inflammatorysteroid.

24. The folate-targeted dendrimer conjugate of any of clauses 19-23wherein the conjugated anti-inflammatory agent is a drug useful in thetreatment of rheumatoid arthritis or other autoimmune disease includingan antiproliferative, immunomodulator or immunosuppresant agent.

25. A method of treatment of an inflammatory disease in a subject inneed thereof, comprising administering an effective amount of afolate-targeted dendrimer conjugate as described in any of clauses18-24.

26. A pharmaceutical composition comprising a folate-targeted dendrimerconjugate comprising an anti-inflammatory agent as described in any ofclauses 18-24 and further comprising at least one pharmaceuticallyacceptable carrier or excipient.

27. A composition comprising a folate-targeted dendrimer conjugatecomprising an imaging or visualizing agent as a conjugated agent andfurther comprising at least one pharmaceutically acceptable carrier orexcipient.

28. A method of using a folate-targeted dendrimer conjugate comprisingan imaging or visualizing agent as a conjugated agent for diagnosis ormonitoring the treatment of an inflammatory disease in an inflamedtissue region in the body in a subject in need thereof.

29. A kit comprising a composition comprising a folate-targeteddendrimer conjugate comprising an imaging or visualizing agent as aconjugated agent as described in clause 27 and instructions fordiagnosis or monitoring the treatment of an inflammatory disease.

30. A kit comprising a folate-targeted dendrimer conjugate comprising ananti-inflammatory agent as described in clause 18-24, or apharmaceutical composition thereof, as described in clause 26, and animaging agent for the inflammatory condition.

Exemplary preparations of folate-targeted liposomal compositionscomprising an anti-inflammatory agent or an imaging agent as anentrapped agent, as well as exemplary preparations of the correspondingnon-folate-targeted liposomal compositions, are provided below in theexamples.

Arteriosclerosis has traditionally been viewed to simply reflect thedeposition of lipids within the vessel wall of medium-sized and largearteries. Upregulation of cell adhesion molecules facilitates adherenceof leukocytes to the dysfunctional endothelium and their subsequenttransmigration into the vessel wall. Evolving inflammatory reactionresults in the initiation of atherosclerotic plaques. A large number ofrecent studies demonstrate that activated macrophages constitute the keyeffector cells in atherosclerosis; and it was reported that the folatereceptor, FR_(β), the nonepithelial isoform of the folate receptor isexpressed on activated (but not resting) macrophages.

That the instant folate-targeted liposomal composition can deliver itsentrapped agent to inflammation induced activated macrophages isdemonstrated in Imaging Example 1, below. In that example the uptake ofcalcein from a calcein-entrapped folate-targeted liposomal compositionby cells from rat peritoneal fluid in vitro is demonstrated in athioglycolate-induced inflammation model using flow cytometry (FIG. 14).The calcein uptake is competitively inhibited by incubation in thepresence of excess folic acid; and the comparable nontargeted (NT)liposomal composition delivers significantly less calcein. Because thedye within the liposomes is quenched at high concentration, thefluorescence is observed only following endocytosis and calceinunloading inside the target cells. Accordingly, such imaging experimentssupport the unloading of therapeutic agents in similar situations.

Apo E mice with the knock out mutation show a marked increase in totalplasma cholesterol levels that are unaffected by age or sex. Fattystreaks in the proximal aorta are found at 3 month of age. The lesionsincrease with age and progress to lesions with less lipid but moreelongated cells, typical of a more advanced stage of pre-atheroscleroticlesions. At 24 weeks of age, mice fed a normal diet show obviousatherosclerotic lesions in the aortic sinus and the ascending aorta. Thenumber of atherosclerotic lesions in the aortic sinus and the ascendingaorta is significantly increased in homozygotes fed an atheroscleroticwestern diet vs a normal diet. Imaging Example 2 demonstrates theincreased uptake of of ³H-cholesterol labeled folate-targeted liposomesin the aorta and hearts of mice fed a high fat western diet compared toa normal diet, indicating the increase in atheroscerotic lesions in thewestern diet animal.

Imaging Examples 3 through 6 demonstrate the ability of folate-targetedliposomal compositions to deliver entrapped imaging agents toatherosclerotic sites in an atherosclerosis model employing Apo E knockout (Apo E KO) mice. Therapeutic Examples 1 and 2 demonstrate theability of betamethasone-entrapped folate-targeted liposomalcompositions to reduce the evidence of atherosclerotic inflammation inApo E KO mice.

In Imaging Example 3, ability of a folate-targeted liposomalcomposition, with the entrapped fluorescent dye DiD, to label theatherosclerotic sites in Apo E KO mice fed a western diet for one weekbefore dosing is demonstrated along with the competitive blocking of thelabeling by pre-administered free folic acid. Imaging of both the wholeanimals (FIG. 2) and the excised aortic arch (FIG. 3) show the labelingof the sites and competitive blocking.

Imaging Example 4 (FIG. 4) provides a similar example in Apo E KO micefed a western diet for four weeks before imaging under four conditions:(A) control with no imaging agent, (B) DiD-entrapped folate-targetedliposomal composition, (C) non-targeted DiD-entrapped liposomalcomposition, and (D) DiD-entrapped folate-targeted liposomal compositionadministered with an excess of folic acid. The images of the mice ofeach of the four treatment groups demonstrate superior uptake of the dyefrom the DiD-entrapped folate-targeted liposomal composition compared tothe non-targeted DiD-entrapped liposomal composition, as well as thecompetitive inhibition of uptake of the dye from the DiD-entrappedfolate-targeted liposomal composition by excess folic acid.

Imaging Example 5 provides an example of an Apo E KO mouse dosed with aniobitridol-entrapped folate-targeted liposomal composition whichdemonstrates the use of an entrapped CT agent for radiographic imaging(FIG. 5).

Imaging Example 6 provides an example using radioisotopic imaging in ApoE KO mice fed a western diet for four weeks before dosing using a⁹⁹Tc-entrapped folate-targeted liposomal composition, adminisered eitheralone or with an excess of folic acid (FIG. 6), demonstrating thecompetitive blockage of uptake of the folate-targeted liposomalcomposition by excess folic acid.

In Therapeutic Example 1, Apo E KO mice were fed a western diet for 3weeks and dosed as (A) a control (Untreated) group which received afolate-targeted liposomal composition which contained only phospatebuffered saline as the entrapped agent, (B) a group (NT Liposome) whichreceived a non-targeted liposomal composition which containedbetamethasone as the entrapped agent, and (C) a group (Fol-Liposome)which received a folate-targeted liposomal composition which containedbetamethasone as the entrapped agent. The mice were imaged using thefolate-receptor targeted, radionuclide conjugate imaging agent EC20(folate-Tc99m) and the biodistribution of EC20 uptake was determined.The reduction in inflammatory cells imaged by EC20 in the Fol Liposomegroup compared to the NT Liposome and Untreated groups is demonstratedby the imaged mice (FIG. 7, top) and excised hearts (FIG. 7, bottom),particulary the heart uptake of EC20 (FIG. 8), and biodistribution (FIG.9).

In Therapeutic Example 2, Apo E KO mice were fed a western diet for 4weeks before dosing as (A) a control (Untreated) group which received afolate-targeted liposomal composition which contained only phospatebuffered saline as the entrapped agent, (B) a group (NT Liposome) whichreceived a non-targeted liposomal composition which containedbetamethasone as the entrapped agent, and (C) a group (Fol Liposome)which received a folate-targeted liposomal composition which containedbetamethasone as the entrapped agent. The mice were imaged using thefolate-receptor targeted, radionuclide conjugate imaging agent EC20(folate-Tc99m) and the biodistribution of EC20 uptake was determined.The reduction in inflammatory cells imaged by EC20 in the Fol Liposomegroup compared to the NT Liposome and Untreated groups is demonstratedby the measures of EC20 uptake in the hearts and the aortas of thegroups FIG. 10 (top); FIG. 10 (bottom) shows the relativebiodistribution of EC20 uptake in the organs of the groups.

Imaging Examples 7 through 9 demonstrate the ability of folate-targetedliposomal compositions to deliver entrapped imaging agents to sites ofinflammation in other tissues and support the use of folate-targetedliposomal compositions comprising an anti-inflammatory agent as anentrapped agent, as disclosed herein, in the treatment of aninflammatory disease, such as in an inflamed tissue region in the body.

Imaging Example 7 demonstrates (FIG. 11) the ability of thefolate-targeted liposomal compositions to deliver entrapped agents toinflamed intestinal tissues, for example in inflammatory bowel disease,such as Crohn's disease or ulcerative colitis, using imaging under thefollowing conditions: (A) DiD-entrapped folate-targeted liposomalcomposition, (B) DiD-entrapped folate-targeted liposomal compositionadministered with an excess of folic acid, and (C) non-targetedDiD-entrapped liposomal composition.

Imaging Example 8 demonstrates (FIG. 12) the ability of thefolate-targeted liposomal compositions to deliver entrapped agents toinflamed arthritic tissues, for example as in rheumatoid arthritis,using imaging under the following conditions: (A) DiD-entrappedfolate-targeted liposomal composition, (B) DiD-entrapped folate-targetedliposomal composition administered with an excess of folic acid, and (C)non-targeted DiD-entrapped liposomal composition.

Imaging Example 8A also demonstrates (FIG. 15) the ability of thefolate-targeted liposomal compositions to deliver entrapped agents toinflamed arthritic tissues, for example as in rheumatoid arthritis,using imaging under the following conditions: a non-targetedDiD-entrapped liposomal composition (NT Liposome), a diseased rat imagedwith a folate-targeted DiD-entrapped liposomal composition(Fol-Liposome), and a healthy rat with a folate-targeted DiD-entrappedliposomal composition (Healthy).

Imaging Example 9 demonstrates (FIG. 13) the ability of thefolate-targeted liposomal compositions to deliver entrapped agents toinflamed skeletal muscle tissues using imaging with (A) a DiD-entrappedfolate-targeted liposomal composition, and (B) a DiD-entrappedfolate-targeted liposomal composition administered with an excess offolic acid.

Imaging Examples 10 and 11 demonstrate (FIG. 16, FIG. 17 and FIG. 18)that the folate-targeted liposomal compositions preferentially deliverentrapped agents to inflammatory tissues as compared to tumors whichexpress a high level of folate receptors on their surface. In addition,the examples demonstrate that the increase in uptake of thefolate-targeted liposomal compositions compared to the non-targetedliposomal compositions is greater in the inflammatory tissues than theincrease in the tumors.

Imaging Example 12 demonstrates uptake of a folate-targeted dendrimerconjugate conjugated to FITC dye and the competitive inhibition of theuptake by excess folic acid in the cells which express the folatereceptor at high levels on the surface.

In Imaging Example 13, ability of a dendrimer conjugate conjugated toCy5.5 dye to label the atherosclerotic sites in Apo E KO mice fed awestern diet before dosing is demonstrated along with a comparison witha non-targeted dendrimer conjugate conjugated to Cy5.5 dye and thecompetitive blocking of the labeling by competition with afolate-targeted dendrimer conjugate which lacks a conjugated dye.Imaging of both the whole animals (FIG. 22) and the excised aortic arch(FIG. 23) show the labeling of the sites and competitive blocking.

Imaging Example 14 demonstrates (FIG. 24) the ability of a dendrimerconjugate to deliver conjugated agents to inflamed intestinal tissues inan ulcerative colitis model using using imaging with a folate-targeteddendrimer conjugate conjugated to Cy5.5 dye, with a non-targeteddendrimer conjugate conjugated to Cy5.5 dye, and in competition with afolate-targeted dendrimer conjugate which lacks a conjugated dye.

The following examples further illustrate specific embodiments of theinvention; however, the following illustrative examples should not beinterpreted in any way to are to limit invention. Abbreviations used inthe examples include the following: Chol, cholesterol; DCC,dicyclohexylcarbodiimide; DiD,1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate;DIPEA, diisopropylethylamine; DMSO, dimethyl sulfoxide; DSPC,distearoyl-phosphatidylcholine; DSPE,distearoylphosphatidylethanolamine; EDC,1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride; mPEG,methyl capped PEG; MS, mass spectrometry; PBS, phosphate bufferedsaline; PEG, poly(ethylene glycol).

EXAMPLES Example Preparations Exemplary Preparation 1 Preparation ofFolate-Targeted Liposomal Compositions Synthesis of Folate-PEG-NH2

Under N₂, Folate-PEG-NH₂ is synthesized using 500 mg of NH₂PEG₃₂₀₀NH₂(PEG-bis amine) with an equimolar quantity of folic acid in 5 mL DMSOcontaining 1 molar equivalent of DCC or EDC and 10 μl pyridine,triethylamine or DIPEA. The reaction mixture is stirred overnight in thedark at room temperature. The reaction is quenched with water. Theby-product urea and the trace amount of unreacted folate and PEG-bisamine are removed by both dialysis and by using a G-15 size exclusioncolumn in deionized water. Folate-PEG-NH₂ is analyzed using HPLC withmonitoring by absorbance at 363 nm.

Synthesis of N-succinyl-DSPE

Under N₂, N-succinyl-DSPE is synthesized by reacting overnight 1.1 molarequivalent of succinic anhydride with 100 mg DSPE in 5 mL of drychloroform or dichloromethane containing 10 μL pyridine, triethylamineor DIPEA. The product N-succinyl-DSPE runs as a spot with a higher Rfvalue (˜0.5) than DSPE and is ninhydrin negative on TLC, usingchloroform/methanol (3:2) as the solvent phase. The structure of theproduct N-succinyl-DSPE is confirmed by MS.

Synthesis of Folate-PEG-DSPE

The synthesis of the folate-PEG-DSPE construct is illustrated above.Under nitrogen, N-succinyl-DSPE is dissolved in chloroform and itscarboxyl-group is activated by reacting with 0.95 molar equivalent ofDCC or EDC. Then an equimolar amount of folate-PEG-DSPE is alsodissolved in the above mentioned reaction mixture and the reaction iscontinued overnight at room temperature. The solvent is then removedunder reduced pressure. The unreacted products are then removed from themixture by membrane dialysis. The product folate-PEG-DSPE is analyzed ona HPLC system chromatography (water/methanol (1.5:1)) using a C-18reverse phase column.

Preparation of Betamethasone-Entrapped Folate-Targeted LiposomalComposition:

126.4 mg of DSPC, 44 mg of cholesterol, 32 mg of mPEG₂₀₀₀DSPE and 1 mgof FolatePEG₃₂₀₀DSPE are weighed and mixed in a 50 mL round bottomflask. The lipid mixture is dissolved in chloroform before evaporatingto dryness in a Rotavapor and kept under vacuum overnight. 200 mg ofbetamethasone phosphate is weighed and dissolved in 6 mL of phosphatebuffered saline. The betamethasone solution is added to the dry lipidfilm, and the mixing is further enhanced at 70 ° C. The mixture issubjected to freeze-thaw cycle 10 times. The liposomal suspension istransferred to an extruder and extruded under nitrogen pressure, 10times each through 200 nm, 100 nm, and 50 nm pore filters, respectively.The mean particle size of the liposome as determined by light scatteringis 60-70 nm.

Preparation of calcein entrapped-folate-targeted liposomal compositions:

In order to prepare calcein entrapped folate-PEG-liposomal compositions,a total mass of 100 mg of the lipid mixture;[DSPC/Chol/mPEG2000-DSPE/folate-PEG-DSPE (56:40:4:0.1) mol %] isdissolved in chloroform and dried to a thin film in a round bottom flaskunder reduced pressure overnight. The lipid mixture is rehydrated in 10mM calcein dissolved in PBS (pH 7.4). The resulting lipid suspension issubjected to 10-15 cycles of freezing and thawing. Then 50 nm and100 nmfolate-PEG-liposomes of different size distributions are generated byextruding the lipid suspension 10 times each through polycarbonatemembranes having pore sizes of sequentially 800 nm, 400 nm, 200 nm and100 nm, followed by 50 nm, if needed, respectively. Unentrapped dye isseparated using membrane dialysis. Particle size is determined byDynamic Light Scattering-DynaPro99. Phosphate assay (Steward assay) isperformed to determined phospholipid concentration.

Preparation of DiD-Entrapped Folate-Targeted Liposomal Composition (1%Dye with Respect to Phospholipid):

126.4 mg of DSPC, 44 mg of cholesterol, 32 mg of mPEG₂₀₀₀DSPE and 1 mgof FolatePEG₃₂₀₀DSPE are weighed and mixed in a 50 mL round bottomflask. The lipid mixture is dissolved in chloroform before evaporatingto dryness in a Rotavapor and kept under vacuum overnight. 15 μl of1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate(DiD) is added in 6 mL of (pH 7.4) phosphate buffered saline. The dyesolution is added to the dry lipid film, and the mixing is furtherenhanced at 70° C. The mixture is subjected to freeze-thaw-vortex cycle10 times. The liposomal suspension is transferred to an extruder andextruded under nitrogen pressure, 10 times each through 200 nm, 100 nm,and 50 nm pore filters, respectively. The mean particle size of theliposome as determined by light scattering is about 60 nm.

Preparation of Iobitridol-Entrapped Folate-Targeted LiposomalCompositions:

Lipid mixture consisting of DSPC/Chol/mPEG₂₀₀₀DSPE/FolPEG₃₂₀₀DSPE in a56:40:4:0.1 molar ratio was dissolved in ethanol at 70° C. The ethanolsolution was then hydrated with iobitridol (350 mg I/mL) for 2 h. Thesuspension of liposomes obtained was submitted to a filtration through400 nm, 200 nm, 100 nm, and 50 nm (10 cycles each) polycarbonatemembranes using an extruder. Liposomes were then dialyzed overnight in a300K Mw cutoff dialysis bag against PBS solution.

Preparation of Chelated ⁹⁹Tc-Entrapped Folate-Targeted LiposomalCompositions by Remote Loading:

(Part 1) Preparation of folate-targeted liposome encapsulatingglutathione.

Folate-targeted liposomes encapsulating glutathione were prepared usinga procedure based on polycarbonate membrane extrusion. Briefy,unilamellar vesicles were prepared fromDSPC/Chol/mPEG₂₀₀₀DSPE/FolPEG₃₂₀₀DSPE (56:40:4:0.1) by thin filmhydration method. Lipids at indicated ratios were dissolved inchloroform, and the solvent was evaporated under vacuum with a stream ofN₂ gas to remove all organic solvent. The resultant film was thenhydrated with 50 mM reduced glutathione in PBS (pH 7.4) at 60° C. withconstant stirring. The suspension of liposomes obtained was submitted toa filtration through 400 nm, 200 nm, 100 nm, and 50 nm (10 cycles each)polycarbonate membranes using an extruder. Untrapped residual reducedglutathione was removed by passing the liposome suspension through aSephadex G50 column.

(Part 2) Radiolabeling of Tc-99m into Liposomes

The commercially available kit of HMPAO, hexamethylpropylene amine oxime(Ceretec®, UK) was labeled with Tc-99m. The kit was reconstituted with15 mCi sodium pertechnetate in 0.9% NaCl solution at room temperaturefor 8 minutes. The 99mTc-HMPAO complex was then mixed with the preformedliposome (part1) and incubated at 37° C. with intermittent vortexing for30 minutes. Uncapsulated 99mTc-HMPAO was removed by gel filtration on aSephadex G50 column using PBS as an eluent.

Exemplary Preparation 2 Preparation of NonTargeted (NT) LiposomalCompositions

For comparison experiments, liposomal compositions are prepared asdisclosed for the preparation of the corresponding folate-targetedliposomal compositions, except the folate-PEG-DSPE conjugate is omitted.

Exemplary Preparation 3 Preparation of Folate-Targeted DendrimerConjugates

An exemplary preparation of a folate-targeted dendrimer conjugateconjugaged to a fluorescent dye (Cy 5.5 NHS or FITC) using a DendritechGeneration 3 (G3) poly(amido amine) (PAMAM), denoted as “Dendrimer,”having a molecular weight of 6,909 Daltons, a measured diameter of 36 Å,and 32 surface primary amino groups follows: On the average, eachresulting dendrimer conjugate contains six folate-PEG residues, ten mPEGresidues and two Cy 5.5 or FITC residues.

Synthesis of Folate-PEG-NH₂

Under N₂, Folate-PEG-COOH is synthesized using 500 mg of NH₂PEGCOOH(Mw′3400) with equimolar quantity of folic acid in 5 mL DMSO containing1 molar equivalent of dicyclohexylcarbodiimide (DCC) or1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and10 μL pyridine, triethylamine or diisopropylethylamine (DIPEA). Thereaction mixture is stirred overnight in the dark at room temp. Thereaction is quenched with H₂O. The trace amount of unreacted folate isalso removed by both dialysis and G-15 size exclusion column in dH₂O.Folate-PEG-COOH is analyzed using HPLC by absorbance at 363 nm.

Synthesis of Folate-PEG-Dendrimer

Under N₂, FolatePEG-Dendrimer is synthesized by reacting overnight 6molar equivalent of FolNHPEGCOOH and EDC (6 eq) with Dendrimer (1 eq) in5 mL of dry DMSO containing 10 μL pyridine, triethylamine or DIPEA. Thetrace amount of unreacted materials is also removed by both dialysis andG-15 size exclusion column in dH₂O. Folate-PEG-Dendrimer is analyzedusing UV/Vis and IR.

Synthesis of Folate-PEG-Dendrimer with mPEG

The synthesis of the pegylated folate—PEG-Dendrimer construct isillustrated above, briefly under nitrogen, mPEG-NHS (10 eq,Mw ˜2000) isdissolved in dry DMSO, and 40 μL pyridine, triethylamine or DIPEA isadded. Then 1 equivalent amount of folate-PEG-Dendrimer is alsodissolved with the above mentioned reaction mixture overnight at roomtemperature. The unreacted mixture is then removed by membrane dialysis,confirmed by UV/Vis and IR.

Loading of FITC or Cy 5.5 into Folate-PEG-Dendrimer:

In order to prepare folate-PEG-Dendrimer loaded with dye (FITC or Cy5.5), 1 equivalent of Folate-PEG-Dendrimer and Cy 5.5 NHS (2 eq) or FITC(2 eq) are dissolved in dry DMSO. 40 μL pyridine, triethylamine or DIPEAis added. The reaction is carried out overnight at room temperature. Theunreacted mixture is then removed by membrane dialysis. The remainingproduct is freeze drid and confirmed by UV/Vis and IR.

Exemplary Preparation 4 Preparation of Non-Targeted Dendrimer Conjugates

For non-targeted dendrimer conjugates, the coupling with FolNHPEGCOOH isomitted.

Exemplary Preparation 5: Preparation of Folate-Targeted DendrimerConjugates without Conjugated Dye

For competition studies, the step of coupling with a dye is omitted toprovide folate-dendrimer conjugates.

THERAPEUTIC EXAMPLES Therapeutic Example 1 Treatment with aBetamethasone-Entrapped Folate-Targeted Liposomal Composition in a3-Week Model of Atherosclerosis in the Apo E Knock-Out Mouse

Three groups of Apo E KO mice (Untreated (A), NT Liposome (B) and FolLiposome (C)) were fed with a western diet for 3 weeks. The Fol LiposomeTreated group was injected i.v., twice per week, with abetamethasone-entrapped folate-targeted liposomal composition. The NTLiposome group was injected similarly with a non-targetedbetamethasone-entrapped liposomal composition. The Untreated group wassimilarly injected with a folate-targeted liposomal composition withonly encapsulated phosphate buffer solution. One day after the lastliposome injection, all groups were injected with the folate-receptortargeted, radionuclide conjugate imaging agent EC20 (folate-Tc99m)before radioisotopic imaging and monitoring of EC20 uptake. The imagesare shown in FIG. 7 (top) of the whole animals and in FIG. 7 (bottom) ofthe hearts of the three groups. The relative EC 20 uptake in the heartfor each of the three groups is shown if FIG. 8. The relative EC 20uptake in various organs is shown in FIG. 9.

Therapeutic Example 2 Treatment with a Betamethasone-EntrappedFolate-Targeted Liposomal Composition in a 4-Week Model ofAtherosclerosis in the Apo E Knock-Out Mouse

Three groups (Untreated (A), NT Liposome (B) and Fol-Liposome (C)) ofApo E KO mice (3 mice per group) were fed a western diet for 4 weeks.The Fol Liposome group was injected i.v. twice per week with afolate-targeted betamethasone-entrapped liposomal composition. The NTLiposome group was similarly injected with a non-targetedbetamethasone-entrapped liposomal composition. The Untreated group wassimilarly injected with a folate-targeted liposomal composition withonly encapsulated phosphate buffer solution. One day after the lastliposome injection, the groups were injected with the folate-receptortargeted, radionuclide conjugate imaging agent EC20 (folate-Tc99m)before radioisotopic imaging and monitoring of EC20 uptake. FIG. 10(top) shows the relative EC20 uptake of the hearts and the aortas of thegroups; FIG. 10 (bottom) shows the relative biodistribution of EC20uptake in the organs of the groups.

IMAGING EXAMPLES Imaging Example 1 Uptake of Calcein from aCalcein-Entrapped Folate-Targeted Liposomal Composition by RatPeritoneal Cells in a Thioglycolate-Induced Inflammation Model

30 g of dehydrated brewer thioglycolate medium powder was dissolved in 1L of deionized water and autoclaved for 25 minutes at 15 pounds pressure(124° C.). The autoclaved medium was kept in the dark under sterileconditions at room temperature for at least 3 months before use.

Thioglycolate recruited macrophages were isolated by peritoneal lavage 3days after intraperitoneal injection of 3.0 mL 3% sterile medium.

After 3 days, the Lewis rats were sacrificed by CO₂ asphyxiation, andthe peritoneal macrophages were harvested by injecting 60 mL of PBS intothe peritoneal cavity. After centrifugal separation of the cellscollected in the PBS, the cells were resuspended into Eppendorf tubeswith FDRPMI 1640 medium. The medium was replaced with fresh mediumcontaining Folate-targeted liposome encapsulating dye (DiD or calcein).For competition studies, excess free folic acid is preintroduced to theperitoneal medium for 30 min before addition of folate targetedliposome. For control, empty folate targeted liposome (PBS) were used.All media were incubated for 2 h at 37° C. . The cells were washed 3times with PBS and resuspended in fresh PBS. The cell bound fluorescencewas analyzed using flow cytometry.

In FIG. 14 are shown the results of the flow cytometry for control andcalcein-entrapped folate-targeted liposomal compositions in the presenceand absence of excess free folic acid.

Imaging Example 2 Labeling and Biodistribution of Atheroscerotic Lesionsin Apo E Knock Out Mice Fed Normal and Western Diets

One set of Apo E KO mice (Normal Chow) was fed a normal diet and one setof Apo E KO mice (Western Diet) was fed a high fat western diet for twoweeks. Each mouse then received a folate-targeted liposome composition(2 mg total phospholipid, i.v) containing 10 μCi ³Hcholesterol-oleoyl-ether. The animals were sacrificed 24 h later, andorgans were removed. Tissue samples were solubilized using Soluene 350(1 mL/100 mg tissue) at 60° C. for 48 h. Tissue solutions were thenbleached to uniform color using 30% hydrogen peroxide, followed by 6 mLHionic-Fluor scintillation cocktail. 1 mL of tissue solution was countedon scintillation counter. The relative biodistribution of uptake of thelabeled liposomes in the aorta and hearts of Normal Chow and WesternDiet mice is shown in FIG. 1 (top); the biodistribution in a number oforgans is shown in FIG. 1 (bottom).

In the following examples, when DiD is the imaging agent, approximately50 nm DiD-entrapped liposomes of DSPC/Chol/mPEG₂₀₀₀DSPE/FolPEG₃₄₀₀DSPE(56:40:4:0.1) were used, along with the corresponding non-targetedliposomes. For competition studies excess 0.1 mM folic acid was used.Administration was i.p.

Imaging Example 3 Imaging with a DiD-Entrapped Folate-Targeted Liposomalcomposition in a 1-week model of atherosclerosis in the Apo E Knock-OutMouse.

Apo E knockout mice are fed with western diet for 1 week. After 7 days,mice received the DiD-entrapped folate-targeted liposomal composition(fol-PEG-liposome-DiD) (0.2 μmol lipid) i.p.(total volume injected: 100μl) in each mouse. For competition studies, mice were pre-administeredfree folic acid (50 μl) before i.p injection of DiD liposome. Imageswere taken 2 h postinjection. The images are shown in FIG. 2 and FIG. 3.

Imaging Example 4 Imaging with a DiD-Entrapped Folate-Targeted LiposomalComposition in a 4-Week Model of Atherosclerosis in the Apo E Knock-OutMouse

Apo E knockout mice are fed with western diet for 4 weeks. DiD-entrappedliposomes were administered. The animals were imaged after 4 hours. Theimages are shown in FIG. 4.

Imaging Example 5 Imaging with Iobitridol-Entrapped Folate-TargetedLiposomal Composition in a Model of Atherosclerosis in the Apo EKnock-Out Mouse

An Apo E KO mouse (7 weeks old) was subjected to i.p. injection of theiobitridol-entrapped folate-targeted liposomal composition describedabove (3.0×10⁻⁶ mol total lipids). The mouse was imaged by X-ray:preinjection, T=2 , 3, 24, 48 hours interval. The images are shown inFIG. 5.

Imaging Example 6 Imaging with a ⁹⁹Tc-Entrapped Folate-TargetedLiposomal Composition in an 8-Week Model of Atherosclerosis in the Apo EKnock-Out Mouse

Two groups of Apo E KO mice were fed with western diet for 8 weeks. Onegroup of Apo E KO mice (competition group) was subjected to 0.1 mM freefolic acid thru i.p. injection. After 1 hour, i.p. injection of the⁹⁹Tc-entrapped folate-targeted liposomal composition was preformed onboth groups of mice. Images were taken 4 hours postinjection. The imagesare shown in FIG. 6.

Imaging Example 7 Imaging with a DiD-Entrapped Folate-Targeted LiposomalComposition in a Murine Intestinal Inflammation (Ulcerative Colitis)Model

Three C57 mice were fed with a 2% dextran sodium sulfate for 7 days.After 7 days, DiD-entrapped liposomes were administered. Intestines wereremoved and imaged after 4 h. The images are shown in FIG. 11.

Imaging Example 8 Imaging with a DiD-Entrapped Folate-Targeted LiposomalComposition in an Adjuvant-Induced Arthritis Model in the Rat

Adjuvant-induced arthritis (AIA) in Lewis rats was induced by injectionof butyricum (0.15 mg) into a hind paw. After 17 days, DiD-entrappedliposomes were administered. The hind paws of the rats were imaged. Theimages are shown in FIG. 12.

Imaging Example 8A Imaging with a DiD-Entrapped Folate-TargetedLiposomal Composition in an Adjuvant-Induced Arthritis Model in the Rat

Adjuvant-induced arthritis (AIA) in Lewis rats was induced by injectionof heat killed mycoplasmia butyricum (0.5 mg), suspended in mineral oil(5 mg/mL), on day 1 into the left hind foot of Lewis rats. Disease wasallowed to progress for 17 days. 100% of animals developed arthritis asevidenced by gross swelling in the injected paw and progressive swellingin all extremities. DiD-entrapped liposomes were administered (i.v.).The hind paws of the rats were imaged after 12 hours. The images areshown in FIG. 15.

Imaging Example 9 Imaging with a DiD-Entrapped Folate-Targeted LiposomalComposition in a Cardiotoxin-Induced Muscle Injury Model in the Rat

Four mice were injected with 100 μL of a 10 μM solution of a cardiotoxin(from Naja mossambica mossambica), and two mice were used as controls.Four days later, the two healthy mice were injected with folate-targetedDiD-entrapped liposomes, as were two mice with cardiotixin-inducedmuscle injury (chosen randomly). The two remaining cardiotoxin-injectedmice (chosen randomly) were treated with non-targeted DiD-entrappedliposome. 300 μg of phospholipid were injected per mouse. The dye usedwas 1% DiD. Images were taken eight hours after a tail vein injection.The images of three of the mice are shown in FIG. 13.

Imaging Example 10 Imaging with a DiD-Entrapped Folate-TargetedLiposomal Composition in a Cardiotoxin-Induced Muscle Injury Model in inMice Implanted with M109 Tumor Cells

One million M109 cells were injected subcutaneously into a shoulder ofBalb-C mice. On day 14 post tumor cell inoculation, the mice wereinjected in the opposite thigh with 100 μL of a 10 μM solution of acardiotoxin (from Naja mossambica mossambica). Four days later, on day18, mice were treated i.v. with a non-targeted DiD-entrapped liposomalcomposition (NT Liposome), with a folate-targeted DiD-entrappedliposomal composition (Fol-Liposome), and with empty folate-targetedliposome (injected empty folate-targeted liposome first, then dosed witha folate-targeted DiD-entrapped liposomal composition) (Competition withFolate Liposome). The mice were imaged 8 hours later. The images of themice are shown in FIG. 17.

Imaging Example 11 Imaging with a DiD-Entrapped Folate-TargetedLiposomal Composition in a Murine Intestinal Inflammation (UlcerativeColitis) Model in Mice Implanted with M109 Tumor Cells

One million M109 cells were injected subcutaneously into a shoulder ofBalb-C mice. On day 11 post tumor cell inoculation, the mice wereadministered 2% dextran sodium sulfate (DSS) for 5 days. The mice werethen treated i.v. with a non-targeted DiD-entrapped liposomalcomposition (NT Liposome), with a folate-targeted DiD-entrappedliposomal composition (Fol-Liposome), and with empty folate-targetedliposome (injected empty folate-targeted liposome first, then dosed witha folate-targeted DiD-entrapped liposomal composition) (Folate LiposomeW Competion). The mice were imaged 8 hours later. The images of the miceare shown in FIG. 18.

Imaging Example 12 Uptake of Folate-Targeted Dendrimer Conjugate byCells which Express the Folate Receptor at High Levels on the Surface(Control) and in the Presence of a Folate-Targeted Dendrimer ConjugateConjugated to FITC dye (FolDendFITC) Alone and in the Presence of ExcessFolic Acid (FolDendFITC w Excess Folic Acid)

Raw cells are plated in each well and treated with 10 μL of theindicated dendrimers (1.0 mg/mL). After 1 hour of incubation, confocalimaging is carried out. The images are shown in FIG. 21.

Imaging Example 13 Imaging in the Apo E Knock-Out Mouse model ofAtherosclerosis with a Folate-Targeted Dendrimer Conjugate Conjugated toCy5.5 dye (FolDend(G3)Cy5.5), with a Non-Targeted Dendrimer ConjugateConjugated to Cy5.5 Dye, and in Competition with a Folate-TargetedDendrimer Conjugate which Lacks a Conjugated Dye (FolDend(G3)Cy5.5 WCompetion)

Apo E knockout mice are fed with western diet are treated with theindicated dendrimer conjugates and imaged 4 hours post injection. Theimages are shown in FIG. 22. The images of the excised aortas are shownin FIG. 23.

Imaging Example 14 Imaging in a Murine Intestinal Inflammation(Ulcerative Colitis) Model with a Folate-Targeted Dendrimer ConjugateConjugated to Cy5.5 Dye (FolDend(G3)Cy5.5), with a Non-TargetedDendrimer Conjugate Conjugated to Cy5.5 dye, and in Competition with aFolate-Targeted Dendrimer Conjugate which Lacks a Conjugated Dye(FolDend(G3)Cy5.5 W Competion)

C57 mice were fed with a 2% dextran sodium sulfate, and the indicateddendrimer conjugates were administered. The mice were imaged after 6 h.The images are shown in FIG. 24.

1. A folate-receptor targeted agent comprising a therapeutic agentuseful for the treatment of inflammatory disease which is afolate-targeted liposomal composition comprising an anti-inflammatoryagent as an entrapped agent wherein the lipid bilayer is primarilycomposed of DSPC/cholesterol with a mol ratio of about 56:40. 2-3.(canceled)
 4. The liposomal composition of claim 1 wherein the lipidbilayer comprises a comprises a folate-targeting conjugate composed of(a) a lipid having a polar head group and a hydrophobic tail, (b) ahydrophilic polymer having a first end and a second end, said polymerattached at its first end to the head group of the lipid, and (c) afolate ligand (Fol) attached to the second end of the polymer, andwherein the folate ligand is a folic acid residue or an analog orderivative thereof, wherein the hydrophilic polymer of the folatetargeting conjugate is a PEG having an average molecular weight of about200-5000 in which the end groups, prior to attachment, are independentlyamino, hydroxy, thiol or carboxy; and wherein the folate targetingconjugate is present at about 0.01 mol % to about 1 mol % in the lipidbilayer. 5-6. (canceled)
 7. The liposomal composition of claim 4 whereinthe lipid bilayer further comprises a hydrophilic coating composed of(a) a lipid having a polar head group and a hydrophobic tail and (b) ahydrophilic polymer having a first end and a second end, said polymerattached at its first end to the head group of the lipid and optionallycapped at its second end, wherein the hydrophilic coating is apolyethylene glycol (PEG), a polylactic acid (PLA), a polyglycolic acid(PGA) or a polyvinyl alcohol (PVA). 8-9. (canceled)
 10. The liposomalcomposition of claim 4 wherein the entrapped agent is ananti-inflammatory steroid.
 11. The liposomal composition of claim 1wherein the entrapped agent is a drug useful in the treatment ofrheumatoid arthritis or other autoimmune disease including anantiproliferative, immunomodulator or immunosuppresant agent.
 12. Amethod of treatment of an inflammatory disease in a subject in needthereof, comprising administering an effective amount of a liposomalcomposition as described in claim
 1. 13. A pharmaceutical compositioncomprising a liposomal composition comprising an anti-inflammatory agentas an entrapped agent as described in claim 1 and further comprising atleast one pharmaceutically acceptable carrier or excipient.
 14. Acomposition comprising a folate-targeted liposomal compositioncomprising an imaging or visualizing agent as an entrapped agent andfurther comprising at least one pharmaceutically acceptable carrier orexcipient, wherein the lipid bilayer of the liposomal composition isprimarily composed of DSPC/cholesterol with a mol ratio of about 56:40and, wherein the hydrophilic polymer of the folate targeting conjugateis a PEG having an average molecular weight of about 200-5000 in whichthe end groups, prior to attachment, are independently amino, hydroxy,thiol or carboxy; and wherein the folate targeting conjugate is presentat about 0.01 mol % to about 1 mol % in the lipid bilayer.
 15. A methodof using a folate-targeted liposomal composition comprising an imagingor visualizing agent as an entrapped agent as described in claim 14 fordiagnosis or monitoring the treatment of an inflammatory disease in aninflamed tissue region in the body in a subject in need thereof.
 16. Akit comprising a folate-targeted liposomal composition comprising animaging or visualizing agent as an entrapped agent as described in claim14 and instructions for diagnosis or monitoring the treatment of aninflammatory disease. 17-18. (canceled)
 19. A folate-targeting dendrimerconjugate comprising a folate-targeted dendrimer conjugated to ananti-inflammatory agent wherein the folate-targeting ligand (Fol) is afolic acid residue or an analog or derivative thereof, a hydrophiliccoating, and a conjugated anti-inflammatory agent; and wherein thefolate targeting conjugate is present at about 12-25% of the dendrimerictermini; the hydrophilic coating residue is present at about 25-40% ofthe dendrimeric termini; and the conjugated to anti-inflammatory agentis present at about 3-13% of the dendrimeric termini.
 20. (canceled) 21.The folate-targeted dendrimer conjugate of claim 19 wherein thehydrophilic coating is a polyethylene glycol (PEG), a polylactic acid(PLA), a polyglycolic acid (PGA) or a polyvinyl alcohol (PVA). 22.(canceled)
 23. The folate-targeted dendrimer conjugate of claim 19wherein the conjugated anti-inflammatory agent is an anti-inflammatorysteroid.
 24. The folate-targeted dendrimer conjugate of claim 19 whereinthe conjugated anti-inflammatory agent is a drug useful in the treatmentof rheumatoid arthritis or other autoimmune disease including anantiproliferative, immunomodulator or immunosuppresant agent.
 25. Amethod of treatment of an inflammatory disease in a subject in needthereof, comprising administering an effective amount of afolate-targeted dendrimer conjugate as described in claim
 19. 26. Apharmaceutical composition comprising a folate-targeted dendrimerconjugate comprising an anti-inflammatory agent as described in claim 19and further comprising at least one pharmaceutically acceptable carrieror excipient.
 27. A composition comprising a folate-targeted dendrimerconjugate comprising an imaging or visualizing agent as a conjugatedagent, wherein the folate-targeting ligand (Fol) is a folic acid residueor an analog or derivative thereof, a hydrophilic coating, and aconjugated anti-inflammatory agent; and wherein the folate targetingconjugate is present at about 12-25% of the dendrimeric termini; thehydrophilic coating residue is present at about 25-40% of thedendrimeric termini; and further comprising at least onepharmaceutically acceptable carrier or excipient.
 28. A method of usinga folate-targeted dendrimer conjugate comprising an imaging orvisualizing agent as a conjugated agent as described in claim 27 fordiagnosis or monitoring the treatment of an inflammatory disease in aninflamed tissue region in the body in a subject in need thereof.
 29. Akit comprising a composition comprising a folate-targeted dendrimerconjugate comprising an imaging or visualizing agent as a conjugatedagent as described in claim 27 and instructions for diagnosis ormonitoring the treatment of an inflammatory disease.
 30. A kitcomprising a folate-targeted dendrimer conjugate comprising ananti-inflammatory agent as described in claim 19, or a pharmaceuticalcomposition thereof, and an imaging agent for the inflammatorycondition.